PAMANTASAN NG LUNGSOD NG MAYNILA

COLLEGE OF MEDICINE
Intramuros, Manila, Philippines

EXPERIMENTS ON PROTEINS
Laboratory Formal Written Report

In Partial Fulfillment of the Requirements in

BIOCHEMISTRY

Presented to:
Department of Biochemistry
PLM College of Medicine

Presented by:
Section 1A
Group # 4

NABUS, Don Karlo R. PAINGCO, Mubarak M. Pascual, Exzel Lens B.

2017- 70001 2017-70069 2017- 70136

RICAFRANCA, John Vincent O. SALVANIA, Rizza Mae V. SAN DIEGO, Kirby D.G.
2017-70045 2017- 70005 2017-70093

November 21, 2017

 I. INTRODUCTION
Our daily lives involve vital biological processes and activities that are carried out
by biomolecules such as hormones, enzymes, structural filaments, and antibodies.
These molecules are called proteins, which generally support the growth and
maintenance of body tissues. Proteins are made up of long chains of amino acid
residues that are folded and arranged according to their specific functions.
Each amino acid has a side chain, that could contain either hydroxylic groups,
sulfur atoms, acidic groups or their amides, basic groups, aromatic rings, imino acid, or
simply an aliphatic group, which basically defines its properties, characteristics, and
function, or the protein as a whole. Amino acids that have aliphatic side chains include
glycine, alanine, valine, leucine, and isoleucine. Serine, threonine, and tyrosine carry
-OH group on their side chain. Tyrosine, along with histidine, phenylalanine, and
tryptophan contain aromatic rings in their side chain. The sulfur-containing amino acids
are cysteine and methionine. Aspartic acid and glutamic acid contains acidic groups
which could be converted to their amide counterpart, asparagine and glutamine,
respectively. Arginine, lysine, and histidine have basic groups in their side chain. Lastly,
proline is an imino acid.
By performing certain tests on proteins, amino acids based on their side chains
present could be detected. In this experiment, albumin, a water-soluble protein found in
plasma and egg white, casein, a phosphoprotein commonly found in mammalian milk
and other dairy products, and gelatin, a colorless and tasteless protein derived from
collagen, were subjected to qualitative tests. These tests include Millon's test,
xanthoproteic test, reduced sulfur test, Hopkins-Cole reaction, biuret test, and heat and
acetic acid test.
Millons test is used to detect the presence of phenol-containing amino acid,
tyrosine, yielding a pink to dark-red coloration on positive result. Xanthoproteic test is
used to detect the presence of aromatic side chain of tyrosine, tryptophan, and
phenylalanine, having a positive result of yellow turning orange upon alkalization.
Reduced sulfur test yields positive result of black deposits on cysteine and cystine.
Hopkins-Cole reaction, also known as glycoxylic acid reaction detects tryptophan
resulting to the formation of a violet ring between 2 layers. Biuret test is a general
protein test that confirms the presence of 2 or more peptide bonds in a solution. Heat
and acetic acid test detects coagulable proteins.

II. OBJECTIVES

III. MATERIALS AND METHODS

Solutions containing casein, albumin and gelatin were separately prepared prior
conducting the experiment. These solutions were subjected to different protein tests to
identify its characteristics and the specific amino acid it contain if possible.
Millons Test
Two to three drops of millons reagent were added to 5 ml of each solution
placed in a test tube. The addition of millons reagent caused white precipitate to form
on each solution. A color change will be observed upon the exposure of the solution to
heat, turning white precipitate into red.
Xanthoproteic test
Xanthoproteic test is a color reaction test to determine the presence of benzene
ring or aromatic ring. In the experiment, one (1) mL of concentrated nitric acid (HNO3)
was added to the solutions of casein, albumin and gelatin. Upon heating, a positive
result should produce a yellow precipitate that will dissolve eventually forming a yellow
solution. The color of the solution deepened upon the addition of NH4OH and NaOH.
Another set-up was made using 0.1% phenol solution as the reagent. Positive
color change was noted in the reaction. This setup was made as a confirmatory test for
xanthoproteic test.
Reduced Sulfur Test
Three test tubes were filled with protein solution with 40% NaOH solution (of 1:1
ratio) were boiled for 2 minutes. The results were then recorded after the addition of 1 to
2 drops of Pb(Ac)2. A color change resulting to black discoloration indicates positive
result.
Glyoxilic Acid Reaction (Hopkins-Cole)
Glyoxilic acid reaction or Hopkin-Coles test is tryptophan specific test. In this
experiment, 1 mL of albumin was mixed with Hopkin-Coles reagent. In an inclined
position, 1 mL of concentrated H2SO4 was allowed to flow on the side of the tube to
form a layer of acid beneath. Formation of purple ring indicates a positive result in this
test.
Biuret Test
On three test tubes, 2 mL of each solution were mixed with 2 mL of 10% NaOH.
After mixing thoroughly, 0.5% CuSO4 was introduced in the solution drop by drop until
purplish color change was achieved.
Coagulation by Heat (Heat and Acetic Acid Test)
Three (3) mL of the solutions were placed in test tubes separately. Place each
solution in a water bath and observe the changes in the solutions. Introduce 2-3 drops
of acetic acid in the solutions then observe the visible results.
 IV. RESULTS
Table 1.1 shows the results of the experiments done. Some of the results
deviated from the theoretical results due to contamination of the reagents used.

Reaction Test Casein Albumin Gelatin

A Red precipitate (+) Red precipitate (+) Red precipitate (+)

B1 Yellow solution (+) Yellow solution (+) Yellow solution (+)

B2 Orange solution (+) Orange solution (+) Orange solution (+)

C Brown solution (-) Dark brown soln (+) No change (-)

D No reaction (-) No reaction (-) No reaction (-)

E Purple solution (+) Purple Solution (+) Purple solution and

precipitate (+)

F1 No reaction (-) Coagulated (+) No reaction (-)

F2 No reaction (-) No Reaction (-) No Reaction (-)

A- Millons Test; B1- Xanthoproteic test - HNO3/Heat; B2- Xanthoproteic test- NH4OH/NaOH; C- Reduced Sulfur test; D-
Hopkin-Coles Test; E- Biurets Test; F1- Coagulation by Heat (Post heating); F2 Coagulation by heat (+Acetic Acid)

Table 1. Summary of results

As depicted in figure X, Millons test showed red precipitate for albumin and
gelatin, while casein showed an overall light red appearance of the solution after
heating. A color change to yellow and orange was seen in xanthoproteic test as shown
in the same figure. For reduced sulfur test, albumin reacted intensely producing a black
discoloration while albumin showed slight change producing a brownish discoloration.
On the other hand, no change was seen in gelatin.
The three solutions reacted negatively with Hopkin-Coles test which may be
caused by the contaminations found within the reagent used. Biurets test on the other
hand, showed a positive result for all the protein solutions. Lastly, as seen in the
coagulation by heat experiment, only the albumin reacted positively after the heating
process and all reacted negatively after the addition of acetic acid in the solution.
A- Millons Test; B1- Xanthoproteic test - HNO3/Heat; B2- Xanthoproteic test- NH4OH/NaOH; C- Reduced Sulfur test; D-
Hopkin-Coles Test; E- Biurets Test; F1- Coagulation by Heat (Post heating); F2 Coagulation by heat (+Acetic Acid); Al- albumin;
Cs- Casein; Gl- Gelatin

Figure X. Actual results of the experiment

V. INTERPRETATION AND DISCUSSION

A. PROTEINS
a. Albumin
b. Casein
c. Gelatin
B. MILLONS TEST
 Millon's test, developed by the French chemist Auguste Nicolas Eugene Millon, is
a chemical test to detect phenol groups in a compound. In the experiment, it was done
to check the presence of phenol-containing amino acid, tyrosine. However, it is not
specific for proteins. Therefore, other tests for proteins, such as the biuret test and the
ninhydrin reaction, must be done to confirm whether the sample which tested positive
for Millons is really a protein.
A positive result to the test is a pink to dark-red colored solution or precipitate.
The Millon reagent is a solution of mercuric and mercurous ions in nitric and nitrous
acids. Therefore, it is highly corrosive and toxic, so caution must be made in handling
the said reagent.
The tyrosine is nitrated to form p-nitrophenol. This then reacts with mercuric ion
to form a complex with the nitrated phenol group, which can be initially seen as a white
precipitate. Then after boiling, it can be seen as red precipitate. Therefore, the red
colored precipitate is due to the mercury salt of nitrated tyrosine.

Figure X. Reaction Involved in Millons Test

Albumin, casein, and gelatin all tested positive for the test because they all
contain tyrosine.

C. XANTHOPROTEIC TEST
Xanthoproteic test is a qualitative test for amino acids with aromatic R groups,
namely tyrosine, tryptophan, and phenylalanine. It is indicated by a color change from
yellow, which is due to xanthoproteic acid formed by nitration of aromatic amino acids,
to orange after alkalinization.
 Figure X. Amino Acids Containing Aromatic R Groups

Aromatic ring of the amino acids is nitrated upon addition of NHO3 giving the
yellow nitrate derivative, xanthoprotein. Then, after addition of a NH4OH and NaOH, an
orange- colored solution was formed due to ionization of the phenolic group.

Figure X. Reaction In Xanthoproteic Test

Tyrosine and tryptophan react more easily than phenylalanine, possibly because
its aromatic group does not react with nitric acid easily since its benzene ring is not
activated. However, when heated further, it is expected to produce positive result as
well.
All samples tested positive because they contained at least one aromatic amino
acid in its structure, as they produced an orange- colored compound after the
experiment. Confirmatory tests may be performed to determine the specific aromatic
amino acid present.
 In application, xanthoproteic test may be done on urine samples to detect the
presence of protein in it. Also, a similar xanthoprotein reaction may be observed when
HNO3 is dropped on the skin or nail which, turns yellow, an indication of the presence of
protein after a period of time.

D. REDUCED SULFUR TEST

E. GLYOXYLIC ACID REACTION
The Glyoxylic Acid Reaction (also known as Hopkins-Cole reaction) is a
biochemical test specific for the presence of the amino acid tryptophan in proteins. The
Hopkins Cole reagent contains glyoxylic acid which is the essential compound for the
reaction. Slow addition of concentrated H2SO4 assists in the hydrolysis of the protein
solution to yield its amino acids. This allows reaction of the free amino acids such as
tryptophan. More specifically, the reagent is sensitive to the presence of the aldehyde
group in the indole ring of tryptophan. The formation of a two-layered solution separated
by a purple ring indicates reaction of glyoxylic acid to tryptophan amino acid. Possibly,
other indole derivatives can yield positive results to the test.

Protein + Glyoxylic Acid + Concentrated H2SO4 Violet ring

The three protein solutions exhibited a lower layer of light brown solution and an
upper layer of cloudy solution. No strong observable purple colored ring was formed
between the junction of the solutions. Theoretically, tryptophan-containing proteins such
as albumin and casein will form a purple-colored ring at the interface between the two
liquid layers. In the experiment, no noticeable ring was produced. This can be explained
by the possible impurity or contamination levels of the sulfuric acid used which
negatively affected the hydrolytic power of the acid. According to Brown (1944) as
stated by Russell (1944), a possible source of interference in glyoxylic acid reactions is
the presence of trace nitrates and nitrites in the sulfuric acid used that affected the
formation of the colored ring.
F. BIURET TEST
The biuret test is a general color reaction test for polypeptides and proteins. It is
an assay that depends on the presence of peptide bonds. The Biuret reagent is solution
containing CuSo4 and KOH. Additionally, the solution contains potassium sodium
tartrate that involved in the stabilization of the coordination complex formed
post-chelation. The reagent does not contain biuret but only reacts with the
peptide-similar bonds found in biuret or carbamylurea.
Upon treatment with the copper sulphate-containing biuret solution in the
presence of an alkali such as NaOH, cupric ions (Cu2+) of the reagent chelate with the
peptide bonds in proteins to form a coordination complex. This reaction can be
observed as a change of the color of the solution to purple or deep blue which can be
read at 545 nm. The reaction below shows the representation of the complex formed by
the chelation of peptide bonds with cupric ions. Furthermore, variations in intensity of
resulting solution can suggest quantity of proteins but is not the primary concern of this
experiment.

Figure X. Reaction of Proteins with Cupric ions of the Biuret Reagent.

In the experiment, a light purple-colored solution were formed for all the protein
set-ups. The color change indicated the presence of proteins due to peptide bonds
reacting with the cupric ions. Albumin, casein and gelatin are all established proteins
thus a positive result was expected. The light purple color indicates a lower amount of
proteins detected and not a. failure of the reaction test. The intensity of the color
produced in the reactions depends on the number of peptide bonds in the solution.

G. COAGULATION BY HEAT
H. INSERT CONCEPT MAP WITH DISCUSSION HERE

Millon- tyrosine
Xanthoproteic - Aromatic

VI. CONCLUSION
Albumin turned positive for all tests. Casein was positive for all tests except
glycoxylic acid test and heat and acetic acid test. Gelatin was positive only for Millons,
xanthoproteic, and biuret tests. Thats all, thank you.

VII. REFERENCES

Gornall, A.G., Bardawill, C.J., & David, M.M. (1949). Determination of Serum Proteins
by Means of the Biuret Reaction. Journal of Biological Chemistry. 177:751.
Rising, M. M., Hicks, J.S. & Moerke, G.A. (1930). The Biuret Reaction. The Journal of
Biological Chemistry. 89:1.
Russell, J.A. (1944). Interference With The Colorimetric Determination of Lactic Acid
(Barker-Summerson Method) by Nitrate and Nitrite Ions. The Journal of
Biological Chemistry. 156:463.
Vasudevan, D.M., Sreeukumari, S. & Vaidyanathan, K. (2013). Textbook of
Biochemistry for Medical Students. 7th ed. Jaypee Brothers Medical Publishers
(P) Ltd. Daryganj, New Delhi 110 002, India.
Xanthoproteic Reaction. Revolvy. Retrieved from
https://www.revolvy.com/main/index.php?s=Xanthoproteic%20reaction